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ligation problems


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#1 biochemistry23

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Posted 15 May 2010 - 04:34 PM

Hi everyone!!!!

So that is my problem! I cut my pet20b vector with BamHI ( 2 hours at 37 degrees). then i heat inactivate it for 10 minutes at 70 degrees and I purify it with the nucleospin kit. after that, I make the ligation with T4 ligase. I use 1ul of ligase and 5ul of the vector (final volume 10ul) and leave it overnight at 16 degrees. the next day i transform DH5A cells, but finally I get no colonies on my plates.....
Any ideas???????????? thank you!!!!

#2 phage434

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Posted 15 May 2010 - 07:25 PM

So, have you run the required control for transformation efficiency? You should be able to transform 10 pg of your uncut vector and get hundreds of colonies.

#3 biochemistry23

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Posted 16 May 2010 - 07:35 AM

So, have you run the required control for transformation efficiency? You should be able to transform 10 pg of your uncut vector and get hundreds of colonies.


Yes, I did the control and I got hundreds of colonies.

#4 phage434

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Posted 16 May 2010 - 08:24 AM

It is hard to believe that if you have high transformation efficiency that you get zero colonies. There is almost always some uncut plasmid, which carries over into a ligation. The usual problem is that you have too many colonies, most of which have no insert (a situation which does not occur here, unless you are neglecting to tell us about what your are doing). I would track this by setting up a RE digest (without enzyme), sampling it, then adding enzyme and cutting, sample again, purify, then sample again, then set up a ligation reaction (without ligase), sample again, add ligase, ligate, and sample again. Transform these different samples and track where you are losing your ability to transform. I'll bet you can't transform the initial sample, but I'd be pleased to be wrong.

#5 fishdoc

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Posted 16 May 2010 - 09:37 AM

Hi everyone!!!!

So that is my problem! I cut my pet20b vector with BamHI ( 2 hours at 37 degrees). then i heat inactivate it for 10 minutes at 70 degrees and I purify it with the nucleospin kit. after that, I make the ligation with T4 ligase. I use 1ul of ligase and 5ul of the vector (final volume 10ul) and leave it overnight at 16 degrees. the next day i transform DH5A cells, but finally I get no colonies on my plates.....
Any ideas???????????? thank you!!!!



Have you checked your concentration of DNA after the nucleospin? Is it possible the kit isn't working properly or a step was missed? Is the antibiotic in the plate correct? Are you sure BamHI is a single cutter? How many ul of transformation did you plate?

Just some of the things that could have gone wrong...




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